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Journal of Lipid Research, Vol. 42, 1687-1698, October 2001
Copyright © 2001 by Lipid Research, Inc.

Original Article

Regulation of sterol regulatory element-binding proteins by cholesterol flux in CaCo-2 cells

F. Jeffrey Fielda, Ella Borna, Shubha Murthya, and Satya N. Mathura
a Department of Internal Medicine, 4-522 JCP, 200 Hawkins Drive, and Veterans Administration, University of Iowa College of Medicine, Iowa City, IA 52242

Correspondence to: F. Jeffrey Field, To whom correspondence should be addressed., f-jeffrey-field{at}uiowa.edu (E-mail)


  ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES

The regulation of sterol regulatory element-binding proteins (SREBP) by cholesterol flux was studied in the intestinal cell line CaCo-2. CaCo-2 cells were incubated for 18 h with micelles containing 5 mM taurocholate and 500 µM oleic acid or micelles containing either 200 µM cholesterol or 150 µM lysophosphatidylcholine. In some incubations, an ACAT inhibitor was added or 25-hydroxycholesterol was substituted for cholesterol. The SREBP-1a transcript was 2-fold more abundant than the SREBP-1c transcript. In cells incubated with micelles containing cholesterol, rates of cholesterol synthesis were decreased and rates of esterification were increased. Cholesterol synthesis was decreased further by ACAT inhibition. Cholesterol influx decreased mRNA levels of SREBP-2, HMG-CoA synthase, HMG-CoA reductase, and fatty acid synthase. ACAT inhibition modestly suppressed gene expression further. Neither SREBP-1a nor SREBP-1c mRNA levels were altered by cholesterol. Despite decreases in gene expression of the sterol-responsive genes by cholesterol, the amounts of precursor and mature forms of SREBP-1 and SREBP-2 were not altered. In contrast, if 25-hydroxycholesterol was substituted for cholesterol, both the precursor and mature forms of SREBP-2 were decreased. The polar sterol decreased the mature form of SREBP-1 but the amount of the precursor form was unchanged. In cells incubated with micelles containing lysophosphatidylcholine, which causes cholesterol to efflux from cells, sterol-responsive gene expression was increased. The amounts of precursor and mature forms of SREBP-1 and SREBP-2, however, were not altered. In contrast, if the cells were depleted of cholesterol by incubating them with lovastatin and cyclodextrin, the mature forms of SREBP-1 and SREBP-2 were increased, as were mRNA levels for the sterol-responsive genes.

The data would suggest that cholesterol influx/efflux regulates mRNA levels of sterol-responsive genes independently of changes in the amount of mature SREBP. In contrast, 25-hydroxycholesterol influx or cholesterol depletion alters the amount of mature SREBP, leading to the regulation of sterol-responsive gene expression. — Field, F. J., E. Born, S. Murthy, and S. N. Mathur. Regulation of sterol regulatory element-binding proteins by cholesterol flux in CaCo-2 cells. J. Lipid Res. 2001. 42: 1687–1698.

Supplementary key words: 3-hydroxy-3-methylglutaryl coenzyme A reductase, 25-hydroxycholesterol, fatty acid synthase, gene expression, micelles


  INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES

At the cellular level, the amount of unesterified cholesterol is tightly controlled. Regulating the amount of cholesterol within a cell is dependent on a rather potent end-product feedback regulation of cholesterol synthesis. This is achieved, in part, by inhibiting or enhancing the expression of genes that encode key regulatory proteins responsible for cholesterol synthesis [see review in ref. (1)]. Mechanisms by which a cell recognizes changes in cholesterol flux and how these changes directly influence transcription rates of target genes that control cholesterol synthesis have been elucidated. Three transcription factors, designated sterol regulatory element-binding proteins 1a, 1c, and 2 (SREBP-1a, -1c, and -2) regulate the transcription of genes that encode enzymes in the biosynthetic pathways of both cholesterol and fatty acids [see review in ref. (2)]. SREBP-1a and SREBP-1c are products of a single gene. SREBP-1a is the more potent activator of genes of both synthetic pathways, whereas SREBP-1c is a weaker transcription factor and tends to be more active in enhancing transcription of genes in the fatty acid synthetic pathway (3). SREBP-2 preferentially enhances the expression of genes of the cholesterol synthetic pathway but can also enhance transcription of genes of fatty acid synthesis as well (4) (5) (6). Precursor, or a membrane form of SREBP, is bound to membranes of the endoplasmic reticulum and nuclear envelope. When a cell is depleted of cholesterol, a two-step proteolytic process releases a mature, or nuclear, form that then enters the nucleus (7). By binding to a 10-bp sterol regulatory element in the promoter of target genes, transcription is enhanced. In times of cholesterol excess, proteolysis of the precursor form is prevented and transcription of these genes is suppressed (8).

Similar to other cells within the body, cells of the small intestine can procure cholesterol from circulating lipoproteins and de novo synthesis (9). Unlike other cells, however, intestinal cells have a continual source of either dietary or biliary cholesterol that can be absorbed from the lumen. Thus, the regulation of cholesterol synthesis in this organ has another layer of complexity that is not found in other cells (9). It might be assumed that the influx of cholesterol from the lumen into an absorptive cell would contribute to the cholesterol pool and, hence, regulate the synthesis of cholesterol in this organ. Although this assumption seems reasonable, several early studies of intact animals have suggested that this may not be the case (10) (11) (12).

In an earlier study performed in the intestinal cell line CaCo-2, changes in apical sterol flux were found to alter the activity of the LDL receptor and HMG-CoA reductase, a key regulatory enzyme in the pathway of cholesterol synthesis (13) (14) (15). Loss of cellular cholesterol caused an increase in gene expression of reductase with an increase in enzyme activity (13) (16). In contrast, sterol influx, particularly cholesterol and low concentrations of 25-hydroxycholesterol, suppressed reductase activity without significantly altering gene expression. It was postulated that posttranscriptional events, including decreased translational efficiency and increased degradation of reductase protein, accounted for most of the regulation of reductase activity (13). The present study was performed to investigate whether changes in cholesterol flux in CaCo-2 cells would regulate the SREBP pathway and hence the regulation of expression of sterol-responsive genes. The results suggest that the influx of cholesterol from bile salt micelles does not significantly alter the proteolytic processing of either SREBP-1 or SREBP-2 despite causing an increase in cholesterol esterification, a decrease in cholesterol synthesis, and a decrease in the expression of SREBP-2 mRNA and other sterol-responsive genes, HMG-CoA reductase, HMG-CoA synthase, and fatty acid synthase. Cholesterol efflux increases mRNA levels of SREBP-2 and sterol-responsive genes but also does not alter SREBP mass. In contrast, if 25-hydroxycholesterol is substituted for cholesterol, sterol-responsive genes and SREBP-2 mRNA levels, both precursor and mature forms of SREBP-2, and the mature form of SREBP-1 are decreased. Cholesterol depletion enhances the amount of the mature forms of SREBP and increases the expression of sterol-responsive genes.


  MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES

[9,10-3H(N)]oleic acid (14 Ci/mmol), [14C]acetate (55 mCi/mmol), and [32P]UTP (3,000 Ci/mmol) were purchased from New England Nuclear (Boston, MA). Protease inhibitors, bovine serum albumin, cholesterol, oleic acid, taurocholic acid, methyl-ß-cyclodextrin, and TriReagent were purchased from Sigma (St. Louis, MO). 25-Hydroxycholesterol was obtained from Steraloids (Newport, RI). MAXIscript T7 and ribonuclease protection assay (RPA) III kits were supplied by Ambion (Austin, TX). Medium 199 was from GIBCO-BRL Life Technologies (Grand Island, NY). Lovastatin was obtained from Merck Research Laboratories (Rahway, NJ). ACAT inhibitor PD 128,042 was a gift from Parke-Davis Pharmaceutical Research Division (Warner-Lambert, Ann Arbor, MI).

Cell culture
CaCo-2 cells were cultured on plastic six-well trays for 14 days as described previously (17).

Stock solutions of taurocholate, cholesterol, and 25-hydroxycholesterol were prepared in 95% ethanol. Oleic acid and lysophosphatidylcholine were dissolved in chloroform. Appropriate amounts of sodium taurocholate and lipids were mixed and solvents were evaporated under a stream of nitrogen. Medium 199 with 10 mM HEPES was then added to obtain the desired concentrations of the lipids. The solution was warmed to 37°C and stirred vigorously before use.

Cholesterol and fatty acid synthesis
CaCo-2 cells were incubated for 22 h at 37°C in 5% CO2 with the appropriate treatment. During the last 4 h of the incubation, 2 µCi of [14C]acetate was added in a small volume of medium 199 to each well. The final concentration of acetate was 0.6 mM. At the end of the incubation, cells were rinsed with medium 199, suspended in 1 ml of 90% methanol containing 0.5 M NaOH, and placed in a boiling water bath for 1 h to saponify lipids. After adding an equal volume of acidic water, the lipids were extracted twice at pH 3.0 with 2 ml of hexanes. To remove residual radiolabeled acetate, the combined hexane extract was washed twice with 1 ml of acidic water. Hexanes were then evaporated under a stream of nitrogen and lipids were dissolved in 0.1 ml of chloroform. Fatty acids and cholesterol were separated by thin-layer chromatography on silica gel plates, using hexanes–diethyl ether–acetic acid–methanol 70:30:1:1 (v/v/v/v) as solvent. Lipids were visualized by exposure to iodine and the radioactivity in fatty acids and cholesterol fractions was determined by scraping the bands and counting in a Packard (Downers Grove, IL) Tricarb 2100-TR liquid scintillation counter.

Lipid synthesis
The sodium salt of oleate was prepared by adding a two times molar excess of NaOH to the oleic acid solution in chloroform. The solvent was evaporated under a stream of nitrogen and the oleate was dissolved in 100 µl of warm water. An appropriate amount of medium 199 containing 10 mM HEPES and bovine serum albumin was added to obtain a fatty acid-to-albumin ratio of 3:1. The final concentration of oleate was 50 µM with 6 µCi of [3H]oleic acid (14 Ci/mmol) per 2 ml per well. During the last 4 h of the 22-h incubation with the respective treatment, the medium was replaced with the labeled oleate-albumin solution. The cells were rinsed with medium 199 and lipids were extracted twice with 1 ml of hexanes–isopropanol–water 3:2:0.1 (v/v/v), pH 3.0. The solvents were evaporated under nitrogen and the lipids were dissolved in 0.1 ml of chloroform. Phospholipids, fatty acids, triacylglycerols, and cholesteryl esters were separated by thin-layer chromatography on silica gel plates, using hexanes–diethyl ether–acetic acid–methanol 70:30:1:1 (v/v/v/v) as solvent. The lipids were visualized by exposure to iodine and the radioactivity in the lipid fractions was determined by scraping the bands and counting.

RT-PCR
DNA-free RNA was extracted from CaCo-2 cells by using a High Pure RNA isolation kit from Boehringer Mannheim (Indianapolis, IN). cDNA were synthesized and amplified with the Thermoscript RT-PCR system from GIBCO-BRL Life Technologies.

RNase protection assays
The plasmid preparations were a gift from M. Brown and J. Goldstein (University of Texas Southwestern Medical School, Dallas, TX). RNase protection assays were performed using antisense RNA probes described in Table 1.


 
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  		Table 1. Plasmids and primers used in RNase protection assays

A T7 promoter was added to the PCR products, using a Lig'nScribe kit from Ambion. To obtain antisense probes, the products were amplified by PCR, using a 5' gene-specific primer and PCR adapter primer. The [32P]UTP-labeled RNA probes were synthesized by using a T7 promoter containing the PCR products or linearized plasmid as template with a MAXIscript in vitro transcription kit from Ambion. The [32P]UTP-labeled RNA probes were purified on denaturing 6% acrylamide-8 M urea gels. Total RNA from CaCo-2 cells was isolated with TriReagent (Sigma). mRNA transcripts were estimated by using an RPA III kit from Ambion. Protected fragments were resolved on denaturing 6% acrylamide-8 M urea gels in Tris-borate-EDTA (TBE) buffer. Eastman Kodak (Rochester, NY) Biomax MS film with TranScreen HE was used to visualize the bands on the gel. In Fig 2 Fig 3 Fig 4 Fig 5, the results are expressed as relative mRNA levels for each gene after estimating the densities of the bands on a given blot, using the NIH Image J program. Except for the probes used to detect SREBP-1a and SREBP-1c transcripts (see below), specific activities of the probes differed. Thus, in Fig 2 Fig 3 Fig 4 Fig 5, comparisons can be made only among or between treatments for a given gene.



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  		Figure 1. Effect of micellar cholesterol on cholesterol, fatty acid, and cholesteryl ester synthesis. CaCo-2 cells were incubated for 22 h with 5 mM taurocholate and 500 µM oleic acid (solid circles), 5 mM taurocholate, 500 µM oleic acid, and 200 µM cholesterol (open circles), or 5 mM taurocholate, 500 µM oleic acid, 200 µM cholesterol, and an ACAT inhibitor (PD 128042, 2 µg/ml) (inverted solid triangles). During the last 4 h of incubation, the incorporation of labeled acetate into unesterified cholesterol (A) and fatty acids (B), or of labeled oleate into cholesteryl esters (C), was estimated. Each point represents the mean ± SE of four separate dishes. * Taurocholate + oleate values are significantly different from taurocholate + oleate + cholesterol values at P < 0.05. {dagger} Taurocholate + oleate values are significantly different from taurocholate + oleate + cholesterol + ACAT inhibitor values at P < 0.05. {ddagger} Taurocholate + oleate + cholesterol values are significantly different from taurocholate + oleate + cholesterol + ACAT inhibitor values at P < 0.05.



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  		Figure 2. Effect of micellar cholesterol on mRNA levels of SREBP-1a, SREBP-1c, SREBP-2, HMG-CoA reductase, HMG-CoA synthase, and fatty acid synthase. After an 18-h incubation with the treatments describe in Fig 1, RNA was isolated from the cells and mRNA levels for the respective genes were estimated by RNase protection assays. Lane 1, taurocholate (TC) and oleic acid (OA); lane 2, TC, OA, and cholesterol; lane 3, TC, OA, cholesterol, and ACAT inhibitor. The data represent means ± SE of nine samples from three individual experiments. A representative autoradiogram of the protected fragments from three separate dishes is shown. * TC + OA values are significantly different from TC + OA + cholesterol values at P < 0.05. {dagger} TC + OA values are significantly different from TC + OA + cholesterol + ACAT inhibitor values at P < 0.05.



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  		Figure 3. Effect of micellar 25-hydroxycholesterol on mRNA levels of SREBP-1a, SREBP-1c, SREBP-2, HMG-CoA reductase, HMG-CoA synthase, and fatty acid synthase. CaCo-2 cells were incubated for 18 h with 5 mM taurocholate and 500 µM oleic acid (open columns) or 5 mM taurocholate, 500 µM oleic acid, and 50 µM of 25-hydroxycholesterol (cross-hatched columns). mRNA levels for the respective genes were estimated by RNase protection assays. The data represent means ± SE of six samples from two individual experiments. § Taurocholate + oleate values are significantly different from taurocholate + oleate +25-hydroxycholesterol values at P < 0.001.



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  		Figure 4. Effect of micellar lysophosphatidylcholine on mRNA levels of SREBP-1a, SREBP-1c, SREBP-2, HMG-CoA reductase, HMG-Co synthase, and fatty acid synthase. CaCo-2 cells were incubated for 18 h with 1 mM taurocholate (open columns) or 1 mM taurocholate and 200 µM lysophosphatidylcholine (cross-hatched columns). mRNA levels for the respective genes were estimated by RNase protection assays. The data represent means ± SE of six samples from two individual experiments. Taurocholate values are significantly different from taurocholate + lysophosphatidylcholine values at * P < 0.05, P < 0.01, and § P < 0.001.



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  		Figure 5. Effect of cholesterol depletion on mRNA levels of SREBP-1a, SREBP-1c, SREBP-2, HMG-CoA reductase, HMG-CoA synthase, and fatty acid synthase. CaCo-2 cells were incubated for 18 h with 2.5% delipidated fetal calf serum containing 25 µM cholesterol and 50 µM 25-hydroxycholesterol (open columns) or 25 µM lovastatin and cyclodextrin (1 mg/ml) (cross-hatched columns). mRNA levels for the respective genes were estimated by RNase protection assays. The data represent mean ± SE of six samples from two individual experiments. § Cholesterol + 25-hydroxycholesterol values are significantly different from lovastatin + cyclodextrin values at P < 0.001.

The protected fragments for SREBP-1a and SREBP-1c contained 14 and 17 [32P]uridine phosphates, respectively. The values obtained from these transcripts have been corrected to reflect the differences in the number of uridines in the protected fragment.

The validity of the RPA and equal loading were established by using the same amount of total RNA per sample, documenting RNA concentration dependence, running triplicates of a sample on each gel, and estimating 18S rRNA as a control (Ambion Technical Bulletin 151) (19) (20) (21) (22) (23). The 18S probe, pTRI RNA 18S, was obtained from Ambion. This probe gives a protected fragment of 80 bp in length. 18S rRNA was estimated in the same tubes as experimental samples, except in the case of SREBP-1a and SREBP-1c, as these probes also give protected fragments of 80 bp.

Immunoblot analysis
Protease inhibitors N-acetyl-leucyl-leucyl-norleucinal (50 µg/ml), 0.5 mM phenylmethylsulfonyl fluoride, pepstatin A (5 µg/ml), leupeptin (10 µg/ml), 1 mM Pefabloc, 10 mM DTT, aprotinin (2 µg/ml), 0.1 mM zinc sulfate, and 0.1 mM copper sulfate were added to all buffers used for preparing cell fractions. All procedures were performed at 4°C. To prepare total membrane fractions, the cells were allowed to swell for 30 min on ice in buffer A [10 mM HEPES-NaOH (pH 7.4), 1.5 mM MgCl2, 10 mM KCl, 1 mM EDTA, 1 mM EGTA] followed by homogenization by passage through a 25-gauge needle five times. The whole homogenate was centrifuged at 100,000 g for 45 min. The resulting pellet containing all cell membranes was suspended in 150–300 µl of buffer B [125 mM Tris (pH 6.0), 160 mM NaCl, 1% Triton X-100] and sonicated for 10 s. This was subsequently centrifuged for 30 min in a microcentrifuge at 19,000 g. The supernatant was designated as the total membrane fraction.

In some experiments nuclear extract and membrane fractions were prepared from CaCo-2 cells by a modification of the procedure described by Wang et al. (8). The cells were allowed to swell for 30 min on ice in buffer A followed by homogenization by passage through a 25-gauge needle 15 times. The homogenate was first centrifuged at 2,000 g to obtain a nuclear pellet followed by centrifugation of the supernatant at 100,000 g for 45 min to isolate a membrane fraction. The nuclear proteins were extracted with 0.1–0.2 ml of buffer C containing 20 mM HEPES-KOH (pH 7.9), 1.5 mM MgCl2, 25% glycerol, 500 mM NaCl, 1 mM EDTA, and 1 mM EGTA. Membrane proteins were extracted with 0.15–0.2 ml of buffer B. Both fractions were sonicated for 10 s followed by centrifugation in a microcentrifuge for 30 min at 19,000 g. Supernatants from these samples were used for analysis of SREBP-1 and SREBP-2 protein mass.

Equal amounts of the protein from each sample were resolved by SDS-PAGE on the same day of preparation and blotted onto a polyvinylidene difluoride membrane. The blot was prepared, dried, stored, and probed by a nonblock technique described by Sadra, Cinek, and Imboden (24). Briefly, the blot was incubated for 30 min with 1x TBS followed by a rinse with water. It was air dried for 15 min followed by three rinses with methanol–water 1:1 (v/v), kept in methanol for 2 min, and air dried. It was stored at room temperature between two sheets of filter paper. All blots were dried for 10 min under vacuum just before probing with antibodies. The blots were incubated for 1 h with primary antibody in TBS containing 1% goat serum, 2% nonfat milk, and 0.1% Triton X-100. They were rinsed with TBS-0.1% Triton X-100 followed by secondary horseradish peroxidase (HRP) antibody for 1 h in TBS containing 1% goat serum, 2% nonfat milk, and 0.1% Triton X-100. Primary polyclonal antibodies raised in rabbits against human SREBP-1 or SREBP-2 were used. Goat anti-rabbit-HRP (A-6154; Sigma) was used as secondary antibody. The HRP signal was detected on blots with SuperSignal West Femto maximum sensitivity substrate (Pierce, Rockford, IL).

Statistical analysis
To determine whether the differences among the three treatments were significant, the data were analyzed at P < 0.05 by one-way ANOVA and by the Student-Newman-Keuls method, using SigmaStat software (SPSS, Chicago, IL). A t-test was used to compare data from two treatments.


  RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES

Effect of micellar cholesterol on cholesterol synthesis and esterification
To address the regulation of cholesterol synthesis by cholesterol influx, CaCo-2 cells were incubated for 22 h with a micellar solution containing 5 mM taurocholate and 500 µM oleic acid with or without 200 µM cholesterol. Although unsaturated fatty acids alone have been shown to regulate SREBP expression (25) (26) (27) (28) (29), oleic acid was included in all micellar solutions because without the fatty acid, the results were inconsistent and changes in cholesterol metabolism were modest. To further expand the intracellular unesterified cholesterol pool, cholesterol esterification was prevented by adding an ACAT inhibitor to some of the incubations. During the last 4 h of the incubation, labeled acetate was added and the incorporation of acetate into cholesterol and fatty acids was estimated ( Fig 1A and Fig B). Compared with cells incubated with micelles alone, the rate of cholesterol synthesis was significantly decreased in cells incubated with micelles containing cholesterol. This was accentuated in cells incubated with micellar cholesterol together with the ACAT inhibitor. In contrast, there was a modest, but significant, increase in fatty acid synthesis in cells incubated with cholesterol and this was enhanced by the presence of the ACAT inhibitor.

To estimate the rate of cholesterol esterification, labeled oleic acid was added during the last 4 h of the incubation and the incorporation of oleic acid into cholesteryl esters was estimated (Fig 1C). In cells incubated with micelles containing cholesterol, the rate of cholesterol esterification was increased more than 2-fold compared with cells incubated with micelles alone. As expected, in cells treated with the ACAT inhibitor, the synthesis of cholesteryl esters was markedly decreased. In data not shown, the incorporation of oleic acid into phospholipids or triacylglycerols was not altered by cholesterol or cholesterol plus the ACAT inhibitor.

It is clear that micellar cholesterol expands cellular cholesterol pools in CaCo-2 cells, leading to downregulation of cholesterol synthesis and increased cholesteryl ester synthesis.

Effect of micellar cholesterol or 25-hydroxycholesterol on gene expression of SREBP, HMG-CoA reductase, HMG-CoA synthase, and fatty acid synthase
To study the regulation of SREBP and sterol-responsive gene expression by cholesterol influx, CaCo-2 cells were incubated for 18 h with micelles alone or micelles containing cholesterol with or without the ACAT inhibitor. After the incubation, mRNA levels were estimated by RNase protection assays for SREBP-1a, SREBP-1c, SREBP-2, HMG-CoA reductase, HMG-CoA synthase, and fatty acid synthase ( Fig 2). 18S rRNA was used as a control to ensure that equal amounts of RNA were analyzed. In CaCo-2 cells, there was 2-fold more of the SREBP-1a transcript than the SREBP-1c transcript. Gene expression of SREBP-1a and SREBP-1c was not altered in cells incubated with micelles containing cholesterol with or without the ACAT inhibitor. In contrast, SREBP-2 gene expression was modestly decreased by micellar cholesterol and this was fur-ther decreased in the presence of the ACAT inhibitor. In cells incubated with micellar cholesterol, mRNA levels of HMG-CoA reductase, HMG-CoA synthase, and fatty acid synthase were approximately half those observed in cells incubated with micelles alone. Again, mRNA levels for the three genes were suppressed further in cells incubated with cholesterol and the ACAT inhibitor.

In another set of experiments, cells were incubated with micelles in which cholesterol was replaced with the polar sterol 25-hydroxycholesterol ( Fig 3). Compared with cells incubated with micelles alone, mRNA levels of HMG-CoA reductase, HMG-CoA synthase, and fatty acid synthase were decreased in cells incubated with micelles containing the hydroxysterol. SREBP-2 gene expression was also decreased by 25-hydroxycholesterol, whereas neither SREBP-1a nor SREBP-1c expression was altered.

Effect of cholesterol deficiency on gene expression of SREBP, HMG-CoA reductase, HMG-CoA synthase, and fatty acid synthase
The results suggested that influx of both cholesterol and hydroxylated cholesterol suppressed mRNA levels of SREBP-2 and the other sterol-responsive genes. To investigate whether cholesterol loss from CaCo-2 cells would regulate expression of these genes, cells were incubated with taurocholate micelles or micelles containing lysophosphatidylcholine. In previous studies, we have shown that micellar lysophosphatidylcholine causes cholesterol to efflux from cells, leading to upregulation of HMG-CoA reductase activity and mRNA levels (16). The results are shown in Fig 4. In cells incubated with micelles containing lysophosphatidylcholine, HMG-CoA reductase, HMG-CoA synthase, and fatty acid synthase mRNA levels were significantly increased. The effects of cholesterol efflux on SREBP gene expression, however, were modest. In cells incubated with lysophosphatidylcholine, SREBP-1a and SREBP-2 mRNA levels were increased 40% and 25%, respectively. Gene expression of SREBP-1c was not altered.

To induce cholesterol depletion, CaCo-2 cells were incubated in lipoprotein-deficient serum containing lovastatin and cyclodextrin. After the incubation, mRNA levels of SREBP and the sterol-responsive genes were estimated and compared with levels estimated in cells incubated with sterols ( Fig 5). Compared with cells incubated with sterols, mRNA levels of SREBP-2, HMG-CoA reductase, HMG-CoA synthase, and fatty acid synthase were significantly increased in cells depleted of cholesterol. In contrast, both SREBP-1 transcripts were unaltered.

Effect of micellar cholesterol or 25-hydroxycholesterol on SREBP mass
To address the effect of micellar cholesterol on SREBP mass, CaCo-2 cells were incubated for 18 h with micelles alone or with micelles containing either cholesterol or 25-hydroxycholesterol. After the incubation, proteins were extracted from total cellular membranes and the amounts of precursor and mature forms of SREBP-1 and SREBP-2 were estimated by immunoblotting. A representative blot is shown in Fig 6. Compared with cells incubated with micelles alone, neither the precursor nor the mature form of either SREBP-1 or SREBP-2 was altered in cells incubated with micelles containing cholesterol. Similar results were observed in cells incubated with cholesterol and the ACAT inhibitor (data not shown). In contrast, the mature forms of both SREBP-1 and SREBP-2 were decreased in cells incubated with micelles containing 25-hydroxycholesterol. Compared with the precursor form of SREBP-1, however, which was not altered by the hydroxylated sterol, the amount of precursor form of SREBP-2 was decreased by 25-hydroxycholesterol.



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  		Figure 6. Effect of micellar cholesterol on (top) SREBP-1 and (bottom) SREBP-2 mass. CaCo-2 cells were incubated for 18 h with 5 mM taurocholate (TC) and 500 µM oleic acid (OA); 5 mM TC, 500 µM OA, and 200 µM cholesterol; or 5 mM TC, 500 µM OA, and 50 µM 25-hydroxycholesterol. After the incubation, the amount of SREBP-1 and SREBP-2 mass was estimated by immunoblotting. An immunoblot of SREBP-1 and SREBP-2 mass estimated in total membranes is shown at the top of each panel. Each blot is representative of three individual experiments showing three separate samples for each treatment. P, Precursor; M, mature. An immunoblot of the mature protein of SREBP-1 and SREBP-2 estimated in nuclear extracts is shown at the bottom of each panel. Each is from a separate experiment showing three separate samples for each treatment. The SREBP bands depicted in each panel are derived from an autoradiogram of a single membrane.

It could be argued that estimating the amount of mature SREBP in total membranes may not reflect the amount within the nucleus, its site of action. This was addressed by estimating the mature form of SREBP in nuclear fractions isolated from CaCo-2 cells after the above-described incubations. The results were similar (Fig 6). The amount of the mature form in the nuclear fraction was unaltered by cholesterol flux but decreased by the influx of the pol ar sterol.

Effect of cholesterol deficiency on SREBP mass
SREBP mass was estimated in CaCo-2 cells that were incubated with micelles alone or with micelles containing lysophosphatidylcholine ( Fig 7). Compared with CaCo-2 cells incubated with micelles alone, the amounts of the precursor and mature (from total and nuclear membranes) forms of SREBP-1 or SREBP-2 were unaltered by lysophosphatidylcholine.



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  		Figure 7. Effect of micellar lysophosphatidylcholine (LPA) on (top) SREBP-1 and (bottom) SREBP-2 mass. The experimental design is described in Fig 4. The amount of SREBP-1 and SREBP-2 mass was estimated by immunoblotting. An immunoblot of SREBP-1 and SREBP-2 mass estimated in total membranes is shown at the top of each panel. Each blot is representative of three individual experiments showing three separate samples for each treatment. P, Precursor; M, mature. An immunoblot of the mature protein of SREBP-1 and SREBP-2 estimated in nuclear extracts is shown at the bottom of each panel. Each is from a separate experiment showing three separate samples for each treatment. The SREBP bands depicted in each panel are derived from an autoradiogram of a single membrane.

Cells were then depleted of cholesterol by incubating them in delipidated fetal calf serum containing lovastatin and cyclodextrin ( Fig 8). After the incubation, nuclear and membrane fractions were isolated. Compared with cells incubated with sterols, there was more mature SREBP-1 and SREBP-2 and less precursor SREBP-1 in cells depleted of cholesterol.



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  		Figure 8. Effect of cholesterol depletion on SREBP-1 and SREBP-2 mass. The experimental design is described in Fig 5. After the incubation, nuclear and membrane fractions were prepared as described in Materials and Methods and the amount of SREBP-1 and SREBP-2 mass was estimated by immunoblotting. The data represent means ± SE of six samples from two individual experiments. A representative immunoblot for three separate samples from each group is shown. Cholesterol + 25-hydroxycholesterol values are significantly different from lovastatin + cyclodextrin values at P < 0.01 and § P < 0.001.


  DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES

Similar to other cell culture models, the SREBP-1c transcript in CaCo-2 cells was a minor transcript compared with SREBP-1a (18). The ratio of SREBP-1c to SREBP-1a in CaCo-2 cells remained constant whether the cells were confluent or subconfluent (3, 5, 7, 10, and 14 days in culture) or whether they were grown on plastic or semipermeable filters (data not shown). In contrast, it has been observed that in organs of intact animals, the SREBP-1c transcript predominates over the SREBP-1a transcript (18). This was also true in cells prepared from hamster intestine although the ratio of SREBP-1c to SREBP-1a transcripts differed in cell populations recovered along the villus axis (30). A reason for the observed difference in the ratio of SREBP-1c to SREBP-1a transcripts between cell culture models and cells of intact organs remains unexplained.

Cholesterol, absorbed by CaCo-2 cells from bile salt micelles, suppressed cholesterol synthesis and enhanced cholesterol esterification. Preventing cholesterol esterification further suppressed the rate of cholesterol synthesis. These results clearly support the notion that cholesterol expanded regulatory pools of intracellular cholesterol, causing the expected changes in cholesterol metabolism. Moreover, the absorbed cholesterol caused a decrease in the expression of all the sterol-responsive genes: HMG-CoA reductase, HMG-CoA synthase, and fatty acid synthase. Neither of these observations was totally unexpected, as in other cell culture models the addition of sterols clearly downregulates cholesterol synthesis and sterol-responsive gene expression [see reviews in refs. (31) and (32)]. What was unexpected, however, was the observation that these changes in gene expression were not associated with changes in the proteolytic processing of the precursor form of SREBP. In cell culture, it has been clearly established that changes in cholesterol flux coordinately regulate the proteolytic processing of both SREBP-1 and SREBP-2 (18) (33) (34). In CaCo-2 cells, this did not occur. Our experimental conditions, however, differed from those of previous reports. To investigate the regulation of SREBP expression by changes in cholesterol flux, our intention was to simulate in cell culture what an intestinal absorptive cell might normally encounter in vivo. In fact, in past studies, we have used a similar experimental design to document the regulation of expression of HMG-CoA reductase by cholesterol flux (13) (14) (15) (16). One difference from other studies is the use of taurocholate micelles to solubilize cholesterol. This, as opposed to cholesterol added in ethanol, will likely increase the amount of cholesterol that crosses the apical membrane. In cell culture, however, bile salts have been shown to alter numerous other cellular metabolic pathways (35) (36) (37) (38) (39) (40) (41). In CaCo-2 cells, for example, taurocholate micelles cause cholesterol to efflux from cells, resulting in increased HMG-CoA reductase activity and gene expression (13). Amounts of the mature forms of SREBP-1 and SREBP-2, however, were not altered in cells incubated with taurocholate micelles alone (F. J. Field, E. Born, S. Murthy, and S. N. Mathur, personal observations). Moreover, even if bile acids prevented in some way the proteolytic processing of SREBP or altered the rate of degradation of the mature protein, it still does not explain the dichotomy observed between the changes in sterol-responsive gene expression and no change in the amount of mature SREBP mass. Another difference in our study was that the effect of unmodified cholesterol was investigated. Cholesterol is rarely used alone to demonstrate regulation of SREBP and/or sterol-responsive gene expression. It is felt that cholesterol is so poorly soluble that it will not adequately cross cell membranes to cause regulation. Others have suggested that unmodified cholesterol is not the regulatory sterol at all, but that oxygenated cholesterol accounts for the observed changes in cholesterol metabolism (42) (43) (44) (45) (46) (47) (48). Thus, in most studies to date, 25-hydroxycholesterol is added together with cholesterol (usually in ethanol) to potentiate the desired regulation (7) (8) (49) (50) (51). This assumes that cholesterol and hydroxylated cholesterol regulate cholesterol metabolism by similar mechanisms. Although difficult to test experimentally, this assumption has not been systematically challenged. The present results would suggest, however, that cholesterol absorbed at the apical membrane of CaCo-2 cells does regulate the rate of cholesterol synthesis and sterol-responsive gene expression. This regulation by cholesterol appears to be independent of a measurable change in the SREBP pathway. Furthermore, the enhancement of expression of sterol-responsive genes by cholesterol efflux in CaCo-2 cells also occurs independently of measurable changes in the amount of mature SREBP.

Do these results with unmodified cholesterol suggest that the SREBP pathway is not functional or is not regulated in CaCo-2 cells? Results from experiments in which micellar cholesterol was replaced with 25-hydroxycholesterol, and from experiments in which cells were depleted of cholesterol by lovastatin/cyclodextrin, strongly argue that the SREBP pathway is operative in CaCo-2 cells. Similar to results in other cell models, the polar sterol coordinately decreased the amount of mature protein of both SREBP, whereas cholesterol depletion increased the amount of the mature form (18) (33) (34). The effect of the hydroxylated sterol on SREBP-1 and SREBP-2 expression, however, differed. The addition of the polar sterol caused a marked decrease in mRNA abundance of SREBP-2 without altering gene expression of either SREBP-1a or SREBP-1c. This has been observed in other cell culture models and makes good sense because the SREBP-2 gene contains a sterol-response element in its promoter, whereas SREBP-1 does not (52) (53). In contrast to SREBP-2, in which both the precursor and mature forms were decreased by 25-hydroxycholesterol, only the mature form of SREBP-1 was decreased by the polar sterol. The data would suggest that in CaCo-2 cells, the amount of the mature form of SREBP-1 is regulated predominantly by proteolytic processing of the precursor form. The amount of the mature form of SREBP-2 is regulated, at least partially if not entirely, at the transcriptional level.

There are other possible explanations for the dichotomy observed between changes in sterol-responsive gene expression and lack of changes in mature SREBP mass by cholesterol flux. It has been suggested that SREBP alone are weak transcription factors and that coregulatory transcriptional factors, which bind to nearby DNA sequences, are required to potentiate the effects of SREBP. Proven coregulators include Sp1 for the LDL receptor gene and CBF/NF-Y for the fatty acid synthase, HMG-CoA synthase, farnesyl-diphosphate synthase, and squalene synthase genes (54) (55) (56) (57) (58). The yin yang 1 protein, in contrast, is a coregulator that acts to repress the transcription of HMG-CoA synthase, LDL receptor, and farnesyl-diphosphate synthase genes (59). Perhaps changes in cholesterol flux alter the amounts or DNA binding of these coregulators. This would, in turn, lead to the suppression or enhancement of sterol-responsive gene expression independently of changes in proteolysis of the precursor SREBP. There is also evidence that SREBP can be modified by phosphorylation that may alter the efficiency of SREBP to bind DNA (60) (61). Thus, in response to changes in cholesterol flux in CaCo-2 cells, either coregulator activation/deactivation or phosphorylation modification of SREBP may play a more important role in regulating the activity of SREBP rather than by proteolysis of the membrane-bound protein. Studies are currently being performed to address both of these possibilities.

The present results in CaCo-2 cells also differed somewhat from results obtained in isolated cells of hamster intestine. Compared with cells prepared from intestinal strips that were depleted of cholesterol by incubation with lovastatin and cyclodextrin, gene expression of SREBP-1c was increased and SREBP-2 gene expression was decreased in cells isolated from strips incubated with sterols (62). Moreover, in a dietary study, compared with SREBP-1c and SREBP-2 gene expression in intestines of hamsters fed a cholesterol-depletion diet, SREBP-1c gene expression was increased and SREBP-2 expression was decreased in intestines of animals fed a cholesterol-enriched diet (30). Thus, in two separate studies performed in hamster intestine, reciprocal regulation of SREBP-1c and SREBP-2 gene expression by cholesterol flux has been demonstrated. A study demonstrating that the SREBP-1c gene is a target of the sterol-binding nuclear hormone receptor, RXR/LXR transcription factor, explains the enhancement of SREBP-1c gene expression by sterols in intestine in vivo (63). It does not, however, explain the failure of sterols to regulate SREBP-1c expression in cell culture. In addition, in the previous dietary study, compared with hamsters fed a normal chow diet, cholesterol feeding did not significantly alter expression of sterol-responsive genes in intestine (30). Obviously, this is in contrast to what was observed in the present study of CaCo-2 cells.

In hamster intestinal cells, the amount of the mature form of both SREBP was in low abundance and difficult to detect. The amount of precursor mass, in contrast, was in abundance (30). This suggests that in intestinal cells of intact animals, cholesterol is in excess and the proteolytic processing of the precursor form is chronically suppressed. In CaCo-2 cells, however, this was not the case. The mature form of both SREBP was in abundance and easy to detect, suggesting either avid proteolysis of the precursor form or suppressed degradation of the mature form. In either case, ample amounts of the mature form would suggest chronic enhancement of transcription of sterol-responsive genes; just the opposite of what would be expected in intestines of intact animals. These differences observed between results obtained in cell culture versus results obtained in intact animals suggest that caution should be used when findings obtained in cell culture are applied to the in vivo situation.

Finally, we cannot completely exclude the possibility that after unmodified cholesterol flux, modest changes in the proteolytic processing of SREBP occurred, leading to small, undetectable changes in the amount of SREBP mature protein and, thus, regulation of gene expression. We find this difficult to believe, however. The absorption of cholesterol caused a 2-fold decrease in sterol-responsive gene expression, not too unlike the extent of suppression of these genes by 25-hydroxycholesterol. If the SREBP pathway were playing a role in regulating gene expression after cholesterol flux, changes of this degree should have been reflected in the amount of mature SREBP protein, similar to that seen with the oxygenated sterol. Perhaps unmodified and hydroxylated cholesterols regulate gene expression and cholesterol biosynthesis by different mechanisms. This would challenge the assumption that hydroxylated sterols act through the same metabolic pathways as cholesterol. Another possibility is that cholesterol is being hydroxylated either during the incubation period or within the cell, which then leads to suppression of sterol-responsive genes. Even if this were true, it still does not explain the dichotomy observed between changes in gene expression without changes in mature SREBP mass after cholesterol flux.


  ACKNOWLEDGMENTS

The authors thank Dr. Jay Horton for helpful discussions throughout this work and Drs. Brown and Goldstein for the SREBP-1a and SREBP-1c plasmids and polyclonal antibodies against SREBP-1 and SREBP-2. This work was supported by the Veterans Administration and by National Institutes of Health grants HL 49264 and 56032.

Manuscript received March 26, 2001; and in revised form May 31, 2001; and in revised form June 29, 2001

Abbreviations: SREBP, sterol regulatory element-binding proteins


  REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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